Our research over the last year had two main directions, as described below. I) Sensing of S. aureus by P. aeruginosa Sensing of ecological competition and an antagonistic response are thought to be common phenomena in multispecies communities. Therefore, we hypothesize that P. aeruginosa can sense the presence of foreign species, and respond by increasing its antimicrobial activity. In a two-species system consisting of P. aeruginosa and S. aureus, we observed that P. aeruginosa could sense exoproducts secreted by S. aureus, and responded by further inhibiting S. aureus growth. Our specific aims of this project are thus to identify the signal molecules produced by S. aureus, the sensing signaling pathway in P. aeruginosa, the molecules underlying the enhanced antimicrobial activity, and the mechanism of action of these molecules. We showed that a previously described peptidoglycan-sensing pathway does not play a significant role in our system. Further, known P. aeruginosa antimicrobial molecules explained only a part of the growth inhibition of S. aureus, suggesting that novel molecules are likely involved in this phenomenon. We used RNAseq to identify the genes and pathways that are differentially regulated in P. aeruginosa upon exposure to S. aureus. Further, using these data, we identified several promoters that could serve as reporters in P. aeruginosa for the sensing of the S. aureus signal. We have recently constructed a fluorescent reporter system, and are now working on using LC-MS on S. aureus exoproducts in combination with the reporter system to identify the S. aureus signal molecules. II) Identify and characterize molecules involved in interactions between S. aureus and P. aeruginosa. P. aeruginosa produces multiple molecules with antimicrobial activity against several other species, but their mode of action, and the pathways by which other bacteria can adapt to them, are not well-defined. We are interested in delineating the effect these molecules have on S. aureus cells, and identifying the adaptive trajectories of S. aureus in their presence. We have evolved multiple populations of S. aureus against the P. aeruginosa redox-active phenazine molecule pyocyanin and have identified mutations in several genes that have not been previously implicated in pyocyanin resistance. We aim to characterize how these mutations confer pyocyanin resistance, test their relevance in clinical isolates, and better describe the cellular effects of pyocyanin on S. aureus. We will similarly study other P. aeruginosa antimicrobial molecules such as the quinolones.